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Early epigenetic changes in KM3-AML
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Figure 1. Gene expression changes in the KMT2A-MLLT3 leukemia model. (A) Volcano plot of differentially expressed genes (red dots are genes with a P value <10-5, and a log2 fold change >3) from RNA-sequencing data of CD34+ cells and CD34+KM3 cells. Highlighted genes include known markers for stem cells, myeloid cells or pre- viously reported biomarkers for KM3-AML. (B) Scatterplot of MN1 and GPR56 expression in approximately 1,000 cases of pediatric AML in the TARGET cohort. Red dots represent patients with KMT2A-rearranged AML. (C) Summary of transcription factor binding motifs found to be enriched in promoters of upregulated genes in (A) with either ShinyGO v0.6134 or MAGICTRICKS.33 (D) Gene expression levels of transcription factor families highlight- ed in (C) in either CD34+ cells (blue) or CD34+KM3 cells (red). (E) High levels of expression of TLR4, S100A8 and S100A9 are seen after introduction of the KMT2A-MLLT3 fusion and are maintained in the model AML. (F) Loss of KDM4B expression through shRNA knockdown in the KMT2A-MLLT3+ cell line (THP-1) induces growth inhibition (*P<0.005, **P<0.001) and (G) reduced expression of S100A8/A9, CEBPb and other cell-cycle related genes. (H) shRNA knockdown of KDM4B in CD34+ cells transduced with the KM3 fusion show gene expression decreases very similar to those in THP-1 cells. FPKM: fragments per kilo- base of exon per million mapped reads
ed knockdown of KDM4B in THP-1 cells showed a signif- icant impact on growth (Figure 1F) and RNA-sequencing from these cells revealed a profound downregulation of S100A8 and S100A9, CEBPb, P2RY2 (a KM3-AML bio- marker17), and other cell cycle regulators (Figure 1G). We next performed the same experiment but with CD34+ cells transduced with the KM3 fusion to validate the changes in our model system. The data showed very sim- ilar gene expression changes in these genes with the exception of CEBPb, although other CEBP family mem- bers were downregulated (Figure 1H). Together, these data support the hypothesis that the early epigenetic changes, at least partially mediated through KDM4B, lead to activation of S100A8/9 as well as other genes required
scriptional program. At the same time, these factors and others upregulated are also involved in immune and inflammatory pathways, suggesting that there may be additional transcriptional programs that are activated and superimposed on the myeloid differentiation that we observe. To determine whether changes in the expression of chromatin-modifying enzymes might be correlated with our RNA-sequencing data, we looked for enzymes whose expression increased at least 2-fold during in vitro culture and whose expression was maintained in both the model and patients’ AML (Online Supplementary Table S2). This analysis highlighted a single gene with this profile, KDM4B, which has been implicated in murine AML, albeit with functional redundancy.37 The shRNA-mediat-
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